EP1328810B1 - Biopuces de format tridimensionnel - Google Patents

Biopuces de format tridimensionnel Download PDF

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EP1328810B1
EP1328810B1 EP01994529A EP01994529A EP1328810B1 EP 1328810 B1 EP1328810 B1 EP 1328810B1 EP 01994529 A EP01994529 A EP 01994529A EP 01994529 A EP01994529 A EP 01994529A EP 1328810 B1 EP1328810 B1 EP 1328810B1
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Prior art keywords
hydrogel
protein
biochip
binding
immobilized
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EP1328810A2 (fr
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Roberto Fagnani
Sonnkap Hahn
Xiaofan Dong
Tony Pircher
Sandra Matsumoto
Pavel Tsinberg
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Biocept Inc
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Biocept Inc
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    • C—CHEMISTRY; METALLURGY
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/5436—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand physically entrapped within the solid phase
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
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    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00614—Delimitation of the attachment areas
    • B01J2219/00617—Delimitation of the attachment areas by chemical means
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    • B01J2219/00603—Making arrays on substantially continuous surfaces
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    • B01J2219/00614—Delimitation of the attachment areas
    • B01J2219/00621—Delimitation of the attachment areas by physical means, e.g. trenches, raised areas
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J2219/00623—Immobilisation or binding
    • B01J2219/00626—Covalent
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    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00623—Immobilisation or binding
    • B01J2219/0063—Other, e.g. van der Waals forces, hydrogen bonding
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00632—Introduction of reactive groups to the surface
    • B01J2219/00637—Introduction of reactive groups to the surface by coating it with another layer
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00639—Making arrays on substantially continuous surfaces the compounds being trapped in or bound to a porous medium
    • B01J2219/00644—Making arrays on substantially continuous surfaces the compounds being trapped in or bound to a porous medium the porous medium being present in discrete locations, e.g. gel pads
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01J2219/0072—Organic compounds
    • B01J2219/00725—Peptides
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
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    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Definitions

  • Microarrays consisting of immobilized nucleic acids, such as DNA, have demonstrated tremendous utility in the high throughput analysis and characterization of biological samples.
  • information is derived regarding the sample's nucleic acid components.
  • biochips may be formed using simple flat plates, e.g. glass slides, or using plates having depressions or wells formed therein.
  • various sequences of nucleic acid oligomers, DNA i.e. single strand DNA, RNA or PNA, are immobilized in a form in which they will then hybridize to complementary sequences from the sample, see U.S. Patent No. 6,242,246 .
  • the derived data is useful in the determination of gene expression and sequence characteristics from a sample.
  • Such data can be a key element in the determination of the genetic base of disease mechanisms and the identification of potential diagnostic and therapeutic targets.
  • Nucleic acid microarray methodology takes advantage of available DNA synthesizers, PCR methods, and developing genetic target information. There would be prospective interest in extending the use of such microarrays to immobilize other binding entities of biological interest, e.g. antibodies or other proteins which might then be used to allow other high throughput analysis using nonhybridization based interactions, which could potentially provide a route to new biological insights not readily be deducible from the use of nucleic acid microarrays.
  • binding entities of biological interest e.g. antibodies or other proteins which might then be used to allow other high throughput analysis using nonhybridization based interactions, which could potentially provide a route to new biological insights not readily be deducible from the use of nucleic acid microarrays.
  • binding entities such as proteins and peptides
  • Immobilization chemistries normally used to anchor materials such as proteins frequently lead to denaturation of these materials due to adherence or direct contact with the solid support surface.
  • attachment chemistries may also be restrictive as a result of multiple, competitively active moieties that are present on many binding entities such as proteins, which is not an issue with nucleic acids, and it is well known that the conformation of many other binding entities, such as proteins, is key to preserving their biological activity and can be easily disrupted by immobilization through multiple moieties on the molecule.
  • a further method used to study protein interactions is the yeast 2-hybrid system.
  • a target protein library is constructed in yeast. This system is designed to express these proteins of interest, with each being linked to a transcription activating region.
  • DNA encoding for a bait protein (or a protein being examined for possible other interacting proteins) is fused to a DNA binding domain and is also expressed in the same library.
  • a reporter gene carrying the corresponding DNA sequence is also included which codes for a detection system, such as a fluorescent protein or a protein with easily detectable biological activities.
  • Another early method commonly employed to study protein interactions is immunoprecipitation of both the capture and the target protein, followed by analysis of the resulting complex using polyacrylamide gel electrophoresis.
  • the capture protein is first incubated with a heterogeneous mixture of proteins allowing it to bind to its target.
  • the resulting complex is then immunoprecipitated using antibodies raised against one protein of the pair, and the complex is separated for analysis by gel electrophoresis and followed by a detection step, e.g. staining by dye.
  • This method is slow and cumbersome, requires significant biochemistry expertise, and likewise does not lend itself to the rapid high throughput analysis of protein interactions.
  • phage display Another method used to study protein interactions is phage display.
  • a library of proteins is expressed on the flagella of certain filamentous phage proteins expressed on the surface of a host bacterium, e.g. E. coli so as to provide an affinity support for such "displayed" proteins.
  • the phage library is then exposed to a number of potential target proteins.
  • the binding of the displayed protein to the target protein allows target identification.
  • This method has a number of limitations, e.g. large molecular weight proteins are difficult to display, and only very few of a phage's filamentous proteins are appropriate for such use.
  • conformational constrictions of the displayed protein have been known to decrease its affinity and consequently affect its ability to bind to its natural ligand.
  • proteins will generally require the use of a method to attach proteins to the surface in a manner so that they may thereafter be used for detection by readily interacting with other materials or molecules of interest, e.g. proteins, commonly referred to as targets.
  • proteins may be bound directly to a surface treated with divalent or trivalent metal ions, such as Cu 2+ or Fe 3+ , to which proteins will naturally bind with varying degrees of affinity.
  • targets then bind to the probes, they can be detected and identified by SELDI TM (surface-enhanced laser desorption/ionization) in combination with a mass spectrometer, as described in US Patent 5,719,060 .
  • SELDI TM surface-enhanced laser desorption/ionization
  • mass spectrometer as described in US Patent 5,719,060 .
  • chemical binding is also used to attach proteins to a substrate surface, while target ligands are labeled with fluorescence tags; thus, any interaction between probes and labeled targets can be detected using a fluorescence-based slide scanner.
  • U.S. Patent No. 6,087,102 describes a method which utilizes a polyacrylamide gel to create individual cells, composed of the electrophoresed protein spots, which can be subsequently crosslinked in situ into the gel to form a biochip. Limitations of the method include difficulties in preparing precise, small cells on the biochip and in potential destructive effects on the capture protein during crosslinking.
  • U.S. Patent No. 5,847,019 describes another approach which utilizes photopolymerizable polymers to form a patterned network layer to fabricate a biochip, using light-reactive free-radical chemistry.
  • the WO 00/65097 patent application which in regard to the current invention may only be considered as state of the art relevant to the question of novelty, pursuant to Article 54(3) and (4) EPC, also concerns methods for preparing a hydrogel biochip.
  • the European patent application number 90118151.1 also concerns improved solid assay support systems where binding agents are immobilized on solid particles which particles are then attached to a pretreated porous support.
  • binding entities such as proteins
  • the present invention provides a biochip in the form of an array of optically clear PEG or PPG-based polymeric microdroplets arranged on a solid substrate, and it provides the capability of forming as many as 1,000 individual reaction cells per square centimeter. Each cell would typically contain at least one binding entity immobilized generally within the volume of the microdroplet or upon its surface. By altering the different binding entities in the cells in the array in a known fashion, an efficient screening of biological samples or compounds for binding interactions or activities can be performed and quantitated. These cells are preferably three-dimensional in form which maximizes the amount of binding entity contained in each and thereby maximizes detection sensitivity.
  • the polymeric microdroplet that forms each cell of such a biochip is selected to provide an environment conducive to retaining the native conformation of the immobilized binding entity, e.g. a protein or peptide.
  • the resultant polymer is preferably a hydrogel that is physically and chemically stable so as to allow sequential washing and other liquid treatment steps and handlings that would be employed in the fabrication and use of a biochip.
  • Polyethylene glycol-based prepolymers which have isocyanate-functional reactive groups are preferably utilized, and when polymerized, a polyethylene glycol hydrogel network is formed, extended and crosslinked by urethane linkages.
  • the prepolymer After initiating the polymerization reaction, the prepolymer is microspotted onto a biochip substrate and allowed to fully polymerize, forming an array of three-dimensional reaction cells.
  • the particular polymer chemistry for the cell has been described in U.S. Patent No. 6,174,683 and international patent application, WO 00/65097 , which disclose biochips that use nucleic acid oligomers as hybridization capture probes. It has now been found that, as a result of further technical developments, polyethylene glycol, polypropylene glycol, or a copolymer thereof can be effectively employed in the fabrication of three-dimensional biochips suitable for containing a variety of nonhybridization binding entities, including proteins and peptides.
  • the invention provides an optically clear hydrogel biochip as defined in claim 1.
  • the invention provides a method of using a biochip to carry out a biochemical assay as defined in claim 10.
  • the invention provides a method of preparing an optically clear isocyanate-functional hydrogel biochip as defined in claim 11.
  • Hydrogels are a class of polymers that can provide a gel matrix that preferably has adequate pore size and high water content to permit diffusion of molecules in and out of the matrix, an ability to bind to the surface of a glass or the like, sufficient optical transparency in a fully polymerized state to minimize any optical interference with fluorescent tags, good structural integrity when fully polymerized, and adequate shelf life for normal research and clinical use.
  • Hydrogels are hydrophilic network polymers which are glassy in the dehydrated state and swell in the presence of water to form an elastic gel. Isocyanate-functional hydrogels possess a number of characteristics that can be used to advantage for the immobilization of nonhybridization binding entities, e.g. proteins.
  • isocyanate-functional hydrogels are meant organic polymers that are capped with isocyanate groups that will function to carry out a desired further polymerization and also covalently bind proteins or the like, or intermediates that in turn attach proteins.
  • polyurethane polymers which are well known in the art and can be formed by reactions between diisocyanates and polyether or polyester polyols, can provide suitable hydrogels for this purpose.
  • Prepolymers are preferably used as a starting material to form biochips using these isocyanate-functional hydrogels, and preferably these prepolymers are formulated to provide hydrated polyurethane, polyurea-urethane and/or polyurea polymeric gels.
  • Hydrogel polymers have been prepared from various prepolymers and used for a wide variety of other applications. Typically, hydrogels are formed by polymerizing a hydrophilic monomer in an aqueous solution under conditions such that a lightly crosslinked prepolymer is initially formed having a three-dimensional polymeric network which gels in concentrated form. Polyurethane hydrogels are formed by polymerization of isocyanate-end-capped prepolymers by the creation of urea and urethane linkages.
  • Suitable isocyanate-functional prepolymers are often prepared from relatively high molecular weight polyoxyalkylene diols or polyols which are reacted with bifunctional or multi-functional isocyanate compounds.
  • the preferred prepolymers are made from polyoxyalkylene diols or polyols that may comprise homopolymers of ethylene oxide units or block or random copolymers containing mixtures of ethylene oxide units and propylene oxide or butylene oxide units. In the case of block or random copolymers, at least 75% of the units should preferably be ethylene oxide units.
  • homopolymers of polypropylene oxide may also, but less preferably, be employed.
  • the polyoxyalkylene diol or polyol molecular weight is preferably from 2,000 to 30,000, and more preferably from 5,000 to 30,000.
  • Suitable prepolymers may be prepared by reacting selected polyoxyalkylene diols or polyols with polyisocyanate, e.g. at an isocyanate-to-hydroxyl ratio of about 1.2 to about 2.2 so that essentially all of the hydroxyl groups are capped with polyisocyanate.
  • the isocyanate-functional prepolymer preferably should contain active isocyanates in an amount of about 0.1 meq/g to about 1.2 meq/g, and preferably about 0.2 meq/g to about 0.8 meq/g.
  • a fairly low molecular weight prepolymer e.g. less than 3,000 MW, should preferably contain a relatively high isocyanate content (about 1 meq/g or higher).
  • the polymerization rate of such a prepolymer should be controlled so as not to polymerize too rapidly to effectively microspot, and in this respect, high molecular weight prepolymers containing a relatively low isocyanate content are generally preferred.
  • Such high molecular weight prepolymers are often prepared by either of two general methods, but others as known in the art can also be used: (1) a polyol (triol or higher) having a molecular weight of at least 2000, is reacted with a polyisocyanate such as isophorone diisocyanate, or (2) a diol having a molecular weight of at least 2000 is reacted with a polyisocyanate and a cross-linking agent, such as glycerol, trimethylolpropane, trimethylolethane, triethanolamine or an organic triamine.
  • a polyisocyanate such as isophorone diisocyanate
  • a cross-linking agent such as glycerol, trimethylolpropane, trimethylolethane, triethanolamine or an organic triamine.
  • Aromatic, aliphatic or cycloaliphatic polyisocyanates may be used.
  • High molecular weight aliphatic isocyanate-capped prepolymers typically gel to a hydrated polymer state in about 20 to 90 minutes, whereas prepolymers capped with aromatic polyisocyanates gel much more rapidly.
  • bi- and multi-functional isocyanates are as follows: toluene-2,4-diisocyanate, toluene-2,6-diisocyanate, isophorone diisocyanate, ethylene diisocyanate, ethylidene diisocyanate, propylene-1,2-diisocyanate, cyclobexylene-1,2-diisocyanate, cylohexylene-1,4-diisocyanate,-phenylene diisocyanate, 3,3"-diphenyl-4,4"-biphenylene diisocyanate, 1,6-hexamethylene diisocyanate, 1,4-tetramethylene diisocyanate, 1,10-decamethylene diisocyanate, cumene-2,4-diisocyanate, 1,5-naphtalene diisocyanate, methylene dicyclohexyl diisocyanate, 1,4-cyclohexyl di
  • Capping of the selected diols or polyols with polyisocyanates to form prepolymers may be effected using stoichiometric amounts of reactants.
  • the isocyanate-to-hydroxyl group ratio may vary as known in this art but should preferably be about 1 to about 3, and more preferably about 1.2 to about 2.2.
  • the capping reaction may be carried out using any suitable conditions, such as at about 20° to about 150°C, under dry nitrogen, for about 2 hours to about 14 days, and preferably in the absence of a catalyst.
  • the preferred temperature is about 60° ° to 100°C, and the reaction terminates when the isocyanate concentration approximates theoretical values.
  • Preferred prepolymers include polyethylene glycol that is end-capped with toluene diisocyanate: a copolymer of ethylene oxide and propylene oxide (optionally with trimethylolpropane) and toluene diisocyanate; toluene diisocyanate-polyethylene glycol-trimethylopropane, methylene diisocyanate-methylene homopolymer; polymeric methylene diisocyanate-polyethylene glycol; polymer of ethylene oxide-propylene oxide-trimethylolpropane and isophorone diisocyanate, and polyethylene glycol trilactate and toluene diisocyanates.
  • Suitable prepolymers of the above types are available from Hampshire Chemical Corp.
  • HYPOL PreMA ® G-50, HYPOL ® 2000, HYPOL ® 3000, HYPOL ® 4000 and HYPOL ® 5000 which formulations generally include copolymers of polyethylene oxide and a minor amount of polypropylene oxide.
  • the main chain of the hydrogel polymer is preferably comprised of polyethylene glycol, polypropylene glycol, or a copolymer of polyethylene glycol and polypropylene glycol. While not to be constrained by any theoretical mechanism, it is believed that the non-ionic, hydrophilic properties of polyethylene glycol and polypropylene glycol hydrogels provide for low levels of non-specific binding of analyte to the hydrogel and also for good compatibility with the immobilized biomolecules so as to maintain native conformation and bioreactivity thereof. Isocyanate-functional hydrogels advantageously absorb large quantities of liquid quickly and in a relatively uniform manner such that the basic overall shape of the gel material is maintained.
  • Polyurethane-based isocyanate-functional hydrogels of this general type are described in U.S. Patent Nos. 3,939,123 (Mathews, et al. ), 4,110,286 (Vandegaer, et al. ) and 4,098,645 (Hartdegan, et al. ). Such polyurethane-based hydrogels have been extensively used as surface coatings and to form flexible or rigid foams; they have also been used to form foams for enzyme reactor systems.
  • biochips are made using an isocyanate-functional hydrogel that is based on a diol or triol of a high molecular weight polyethylene oxide, polypropylene oxide, or a copolymer of polyethylene oxide and polypropylene oxide, capped with water-active diisocyanates, and which may be optionally lightly crosslinked with a suitable cross-linker. It is preferred that the quantity of active isocyanates present in the prepolymer is predictable, for example preferably between about 0.1 and about 1 meq/g, but more preferably not greater than about 0.8 meq/g.
  • diisocyanates include aromatic-based diisocyanates, such as toluene diisocyanate or methylene diphenyl-isocyanate, as well as aliphatic diisocyanates, such as isophorone diisocyanate.
  • aromatic-based diisocyanates such as toluene diisocyanate or methylene diphenyl-isocyanate
  • aliphatic diisocyanates such as isophorone diisocyanate.
  • about 15% to about 5% of the reactive isocyanates in the polymer are used to provide sites for immobilizing binding entities, and more preferably 10% or less of the reactive isocyanates in the prepolymer are employed to immobilized binding entities.
  • the polymerization of the prepolymer for biochip creation which may be preformulated in a water-miscible organic solvent, takes place by the formation of urea linkages which occur upon the simple addition of water.
  • a binding entity can be directly or indirectly immobilized in each cell or microspot either prior to, during, or after polymerization of the cell material.
  • Indirect immobilization contemplates the employment of an intermediate agent that is first linked to the hydrogel and possibly a second intermediate agent that is, in turn, linked to it.
  • a first or primary intermediate agent that is encapsulated into the hydrogel matrix might be an antibody directed against calmodulin.
  • the calmodulin serves as a second intermediate agent as it is utilized, in turn, to sequester calmodulin-binding-proteins, such as the calcium/calmodulin dependent kinase IL
  • This approach to attaching CaM kinase II (as it is commonly referred to) to the hydrogel provides a gentle way of anchoring the protein via a naturally occurring binding motif, i.e. through the calmodulin protein.
  • the CaM kinase II is now free to probe analyte solutions, for example for the purpose of examining regulatory events on the CaM kinase II (phosphorylation, dephosphorylation) or for searching for possible docking proteins or other intracellular trafficking proteins.
  • Alternative ways of anchoring the CaM kinase II may lead to its loss of function or to other deleterious effects.
  • binding entity is used to refer to material capable of interacting in a specific fashion with one or more target molecules to physically sequester them by a mechanism other than hybridization.
  • nonhybridization binding entities include, but are not limited to, biological material, such as proteins including receptors, peptides, enzymes, enzyme inhibitors, enzyme substrates, immunoglobulins, e.g. antibodies, antigens, lectins, modified proteins, modified peptides, double-stranded DNA, biogenic amines and complex carbohydrates; they may also include synthetic molecules, e.g. drugs and synthetic ligands, designed to have specific binding activity of this type.
  • modified proteins or polypeptides are meant those proteins or peptides having one or more amino acids within the molecule altered by the addition of new chemical moieties, the removal of existing chemical moieties or some combination of both removal and addition. This alteration may include both natural and synthetic modifications. Natural modifications include, but are not limited to, phosphorylation, sulfation, glycosylation, nucleotide addition, and lipidation. Synthetic modifications include, but are not limited to, chemical linkers to facilitate binding to the hydrogel, and the addition of fluorescent dyes, microstructures, nanostructures, e.g. quantum dots, or other synthetic materials. In addition, modification may include the removal of existing functional moieties, e.g.
  • complex carbohydrates include, but are not limited to, natural and synthetic linear and branched oligosaccharides, modified polysaccharides, e.g. glycolipids, peptidoglycans, glycosaminoglycans or acetylated species, as well as heterologous oligosaccharides, e.g. N-acetylglucosamine or sulfated species. Also included are synthetic modifications thereof, such as the addition of molecules such as drugs, ligands, dyes or other agents useful for the purpose of screening and quantitation.
  • Naturally-occurring complex carbohydrates are chitin, hyaluronic acid, keratan sulfate, chondroitan sulfate, heparin, cellulose and carbohydrate moieties found on modified protein such as albumin and IgG. Combinations of two or more of such entities might be immobilized at some locations on the microchip array, which combinations might be added as one mixture of two entities or may be added serially.
  • the interaction between the immobilized entity and the target as sequestering or nonhybridization binding, it is meant that two or more molecules adhere or bind together in a specific and selective fashion, typically by covalent or non-covalent bonds (e.g., by van-der-Waals forces and/or ionic interactions).
  • the specific target can be a simple molecule that may be present in a complex mixture of biological or synthetic materials.
  • the sequestering or binding may be of an extended nature, e.g. covalent modification or antibody-antigen interaction, or it may be transient, e.g. as would occur during a phosphorylation event.
  • DNA binding entities include, but are not limited to, synthetic and natural double-stranded polymers of deoxyribonucleotides, synthetic and natural poly ribonucleotides, aptamers, and synthetic polynucleotides having one or more modified or non-naturally occurring chemical entity.
  • This use of DNA as a binding/sequestering agent is in contrast to conventional nucleotide hybridization arrays which typically employ single strands of DNA (oligonucleotides or cDNA) to which target DNA hybridizes.
  • Double-stranded DNA might be employed to interact with (as opposed to hybridizing) a suitable biomolecule, such as a DNA binding protein, a transcription factor, e.g.
  • estrogen receptor or a synthetic drug or molecule, so as to bind or sequester that biomolecule.
  • general transcription factors such as TBP or SP1
  • gene specific transcription factors such as nuclear hormone receptors
  • Aptamers are described in U.S. Patent No. 5,840,867 where they are indicated to function much like monoclonal antibodies.
  • an embodiment may employ an initial binding entity which is physically co-polymerized within the gel matrix, e.g. select antibodies or other selective binding agents, e.g. aptamers, wherein one or more different antibodies would be immobilized in each cell of an array.
  • an initial binding entity which is physically co-polymerized within the gel matrix, e.g. select antibodies or other selective binding agents, e.g. aptamers, wherein one or more different antibodies would be immobilized in each cell of an array.
  • select antibodies or other selective binding agents e.g. aptamers
  • an antibody against a specific antigen is immobilized within each gel microspot during polymerization; thereafter, specific protein or peptide antigens are provided to bind to each of the cognate antibodies by exposing a mixture of protein or peptide antigens to such an array.
  • an intermediate antibody array is to self-sort a complex mixture of proteins from cell extracts without requiring individual isolation of each protein. Such an array thus formed might then be employed to assess what the effect would be on each site of exposure to an added protein kinase or other protein-modifying moiety. This concept might be extended to examine whether such modifying activities would be influenced by drugs or other added chemical compounds.
  • binding entities may be located or anchored within a cell of a biochip array after polymerization through the use of intermediate agents that will be initially immobilized.
  • a suitable intermediate such as Protein A
  • a desired immunoglobin capture agent is bound to the immobilized Protein A by controlled exposure to the immunoglobin in solution.
  • the initial immobilized binding entities may be subsequently modified.
  • modifications may include (a) biological modifications, e.g. phosphorylation, glycosylation, acetylation, methylation, ubiquitination, lipid modification and ADP-ribosylation, or (b) non-biological modifications, e.g. fluorescent dye modification, biotinylation, alkylation and abnormal residue incorporation, as well as by conjugation with another protein or enzyme to yield an altered final form of the array.
  • double-stranded nucleic acid oligonucleotides are immobilized during polymerization; thereafter, the desired proteins are bound to such nucleic acids by nucleic acid sequence-specific protein interaction, to produce a self-assembled protein-nucleic acid complex array.
  • the binding entity By first reacting the prepolymer with the binding entity in an aprotic solvent, the binding entity is effectively immobilized onto the prepolymer, and this procedure may have a number of advantages in the preparation of the hydrogel. It may help to subsequently generate a homogeneous solution of the prepolymer in water, and it may also serve to slow down the generation of carbon dioxide during the polymerization step as lowering the viscosity of the polymerizing mixture allows CO 2 to slowly effervesce. In the polymerization of the polyurethane-based hydrogels, for example, some gaseous carbon dioxide is generated by the reaction of water with the isocyanate groups of the hydrogel prepolymer. Such reaction is illustrated in FIGS.
  • a further advantage of derivatizing the hydrogel in an aprotic solvent is an enhancement of the optical clarity or transparency of the hydrogel by minimizing any precipitation of the prepolymer.
  • Another way to achieve slow polymerization of the gel and, therefore, slow generation of CO 2 , to assure continuous and transparent gel matrix is to maintain the ratio of aprotic solvent to water at at least about 0.25 to 1 and preferably higher, e.g. 0.3 - 0.35 to 1.
  • Derivatization and polymerization of the hydrogel is generally accomplished in about thirty minutes of such ratios.
  • the quantity of binding entity bound to the prepolymer of any cells is easily adjusted by simply varying the amount of binding entity added to the reaction (for example, from about 10 fmol up to about 1 pmol of protein for each microdroplet), thereby permitting close control over the amount of binding entities immobilized within each hydrogel microdroplet.
  • Ease of diffusion of a prospective target molecule or other secondary binding entity through the gel to interact with an intermediate or primary binding entity immobilized within the gel matrix will be determined, in some part, by the percentage of hydrogel prepolymer in the solution that is employed.
  • the '683 patent describes the employment of 5% solution of prepolymer for formulating hydrogel droplets; however, at 5% level, diffusion of larger molecules, such as proteins, into the polymerized hydrogel is slower than desired. It has now been found that a lower percentage of prepolymer, e.g. 3.5% is preferred to facilitate passage of larger biomolecules into the hydrogel. However, below about a 3% prepolymer solution, the resultant gel may lack sufficient structural integrity and/or adequate polymerization to be useful.
  • the preferred range of polymer is felt to be between 3% and 5%.
  • Other applications and uses such as those for examining molecules smaller in size than a typical antibody, e.g. IgG (or larger, e.g. when the gel encloses or anchors microspheres), may respectively employ a higher or lower percentage of polymer in the solution.
  • hydrogel When the hydrogel is first derivatized with protein and then deposited onto the solid substrate, after initiation and before completion of polymerization thereof, delivery may be accomplished by any convenient method; for example, a conventional microspotting machine which deposits gel to form an array of microdroplets may be used. While such a gel may inherently non-covalently attach to some substrates, a substrate surface is generally derivatized prior to addition of the hydrogel to achieve firm attachment of the gel to the substrate. For example, in one preferred embodiment where glass is used as the substrate, the glass is derivatized with amine prior to deposit of the polymerizing hydrogel.
  • partial initial blocking of the binding entity may be preferred to maximize efficient immobilization of the binding entity.
  • the reactivity of the isocyanate prepolymer with certain chemical moieties that a particular binding entity may include, e.g. primary amines, may result in excess crosslinking between the binding entity and polymer, and such may lead to denaturation of the binding entity or may lower its binding affinity for its target compound Such might be avoided or limited by protecting at least some of these moieties during polymerization; deprotection after polymerization would then return the functionality and utility of the binding entity within the array, i.e. de-blocking after polymerization would allow the binding entity to assume its native conformation.
  • Such blocking/de-blocking may be accomplished by either covalent or non-covalent means.
  • an antigen recognition site susceptible of becoming crosslinked to the polymer is incubated with an uncross-linkable peptide (or other epitope mimic) prior to mixing with the prepolymer.
  • an uncross-linkable peptide or other epitope mimic
  • Such peptide or epitope mimic would protect the antigen recognition site from conjugation with the reactive isocyanate groups during the polymerization process.
  • such peptide would be released from the antibody, e.g., by brief exposure to acid, pH 3.0, thus re-exposing the antigen recognition site of the antibody.
  • Similar mechanics can be employed to protect select sulfhydryl moieties or amines on binding entities; these could use well know reversible chemical derivatization to protect these functionalities while polymerization proceeds.
  • Non-binding additives may be optionally included in the prepolymer mix for stabilization of the binding entities. These additives include, but are not limited to, glycerol, Ficoll, and ethylene glycol as well as saccharides such as mannitol, sucrose and trehalose.
  • non-specific (non-binding) proteins such as bovine serum albumin
  • bovine serum albumin can also be employed to aid in the activity of entities, e.g., proteins, when it is desired to limit the extent of crosslinking to the hydrogel.
  • additives are to employ materials that create zones or domains within the polymerizing hydrogel. Upon completion of polymerization, these materials are either dissolved or diffused away in aqueous solution, leaving larger pores, vacuoles or channels within the hydrogel polymer than would otherwise be present in the absence of these materials. The presence of such larger pores creates a larger surface area on and within the hydrogel cell, providing an increased capacity for binding biomolecules or the like that might be too large to easily diffuse through the hydrogel matrix.
  • the hydrogel polymer is also suitable for other binding entities, including, but not limited to, materials such as synthetic molecules, drugs, non-peptide receptor ligands, mixed organic/inorganic species, e.g. metal porphyrins, and inorganic materials, e.g. zeolites.
  • these entities are used to sequester compounds from solutions based upon specific interactions between the binding entity and the analyte species.
  • these binding entities transiently interact with species in solution. Such is the case when the binding entity serves as a selective substrate for a reactive process, e.g. phosphorylation, methylation, cleavage or other forms of modification.
  • the incorporated materials may be involved in the catalysis of reactions.
  • Such catalytic materials may be useful in bioreactors.
  • an array of different catalytic entities may be used to screen for the most efficient catalytic entity.
  • these hydrogels formulated with such binding entities are useful for a variety of tasks, including, but not limited to, bioassays, materials screening and sensors.
  • a non-biological compound such as a tridentate or tetradentate metal chelating agent, e.g. iminodiacetic acid or nitrilotriacetic acid, having a proper linker of amine-derivatized C 4 -C 8 , is immobilized within the hydrogel as an intermediate binding agent, either prior to or during polymerization as schematically depicted in FIG. 3A .
  • the desired binding entity e.g. a protein, is preferably synthesized or modified so as to have a multiple histidine-containing sequence, e.g.
  • each cell of the biochip can then be immobilized to each cell of the biochip by exposure along with a divalent or trivalent metal ion, such as Cu 2+ or Fe 3+ , so as to allow chelation with such a terminal residue to physically immobilize the protein within the hydrogel by linking to the immobilized chelating agent, as schematically depicted in FIG. 3B .
  • a particular binding entity e.g. a different protein capture agent
  • One advantage of employing an intermediate agent in creating such a polymer microdroplet is the greater assurance that potential denaturation of a particularly susceptible protein is avoided so that the conformation and configuration of the ultimate binding entity protein remains unaltered. Also, fabrication may be simplified by the use of the same chelating agent for creating each microdroplet or cell in a particular protein microarray and then subsequently linking the binding agents thereto.
  • the biochip substrate may consist of a variety of materials and formats which are conducive to automated handling during a binding assay and later detection of target molecules binding to the individual cells.
  • solid flat plates e.g. glass slides
  • plates that have depressions or wells formed therein to hold individual cells may be used.
  • An optically transparent substrate such as glass or clear polystyrene, will allow for transmission light detection through the cells and is convenient for detection modalities using fluorescence or optical absorption. Due to the high binding capacity of three-dimensional hydrogel cells, reflective optical methods are also possible and allow the use of opaque substrates.
  • the use of rigid substrates would allow for precision of alignment during the detection phase of analysis using a biochip, but such may not be necessary if proper alignment is incorporated into the cells to facilitate detection.
  • a flexible format such as a tape or filament
  • a scanning fashion similar to the use of magnetic tape.
  • other biochemical detection methods might alternatively be used, e.g. the detection of radioactive agents.
  • any number of cells can be provided on a biochip, e.g. from 1 to 1000.
  • multiples of 96 cells may be used; for example, 3 84 cells may be provided in an array on a 3 in (7.6 cm) x 5 in (12.7 cm) plate. Although multiple cells are preferably used, a biochip utilizing only a single cell may be satisfactory in certain situations.
  • binding entity it may be preferable to load the binding entity into the hydrogel cell after polymerization of the hydrogel cell.
  • Simple diffusion may be an ineffective tool by which to accomplish this.
  • Small molecules that rapidly diffuse into the hydrogel may, in the course of subsequent use, readily diffuse out of the hydrogel, thereby causing the loss of these binding entities. Therefore, in the case of such readily diffusable agents, e.g. small molecules and peptides, it is preferable to have a mechanism to covalently conjugate such agents to the polymeric matrix after diffusion into the matrix.
  • One preferred means to accomplish this utilizes a moiety suitable for performing crosslinking, e.g. photoactivated or chemical crosslinking reagents, contained either within the polymer as part of its composition or linked to the small molecule diffusing into the polymer.
  • larger binding entities e.g. proteins and large segments of DNA
  • an electric field is applied in such a fashion as to cause the controlled migration of species having a net charge, e.g. proteins, within a solution having a pH value different from the isoelectric point of the protein. This process is termed "electrophoresis". If the hydrogel cell is within the migration path of the charged species, the charged species undergoes an additional force supplied by the applied electric field in addition to passive diffusional forces, thereby accelerating its insertion into the hydrogel cell.
  • An advantage of this electric-field-facilitated diffusion is that these larger binding entities will not readily, passively diffuse out of the hydrogel matrix during subsequent assay steps.
  • the substrate surface not occupied by the hydrogel cells may be treated with agents or materials to reduce subsequent non-specific or non-desired adherence of assay reagents, target molecules or other materials. This is especially useful in those applications where assay reagents may potentially nonspecifically bind to the surface, and thus might substantially reduce the effective concentration of the assay reagents or target molecules in solution. Alternatively, such treatment may be employed to decrease the amount of background signal observed from the surface and thereby increase the effectiveness of the hydrogel cell for assay purposes.
  • Treatments for such exposed surface regions include the application of reagents that react with primary amines which are present as an initial layer or coating on the substrate surface.
  • reagents include, but are not limited to, activated polyethylene glycol polymers having at least one end containing a reactive moiety, e.g. isocyanate, that will covalently bind to a primary amine; and small, non-polymeric molecules functionalized with nucleophile-reactive moieties, such as succinyl esters.
  • standard blocking reagents e.g. bovine serum albumin, customarily employed to reduce background signals as well known to those skilled in the art of molecular biology applications may alternatively be used.
  • the hydrogel microdroplets following polymerization on the substrate, are preferably at least about 30 ⁇ m thick, more preferably at least about 50 ⁇ m thick and most preferably between about 50 ⁇ m and 100 ⁇ m thick.
  • the microdroplets are generally elliptical in shape, as opposed to the square gel cells previously used in some systems. The overall larger size of the gel microdroplets (or cells) permits a significant increase in the quantity of binding entities immobilized therein, thereby reducing the lower detection limit of the biochip and facilitating its use.
  • microdroplets By decreasing the viscosity of the polymer solution and with appropriate modifications to dispensing mechanisms heretofore used for microspotting onto on to a biochip substrate, smaller individual cells can be produced enabling very high-density biochip arrays. If substrates having wells are employed, the microdroplets should be deposited upon the bottoms of the wells.
  • biochip suitable for study of protein-protein interactions is illustrated by binding calmodulin to calcinerine in a calcium-dependent manner.
  • a biochip suitable for protein-DNA interactions is illustrated by the binding of lambda repressor protein to DNA. It should of course be appreciated that these biochips are suitable for antigen-antibody interactions and for other such interactions as mentioned hereinbefore that may not be specifically illustrated in the working examples.
  • the formulation also included 3.5 weight % final concentration HYPOL PreMA ® G-50 hydrogel prepolymer (premixed stock solution containing HYPOL, acetonitrile, N-methyl-2-pyrrolidinone at a w/w/w ratio of 1:3:3, respectively), anti-transferrin (4 mg/ml phosphate buffered saline 1X (PBS), 2 ⁇ l bovine 1gG (50/mg/ml in PBS and 1.25% glycerol).
  • the amount of trehalose was varied from 0 to 10 ⁇ l, corresponding to a final w/v percentage of 0, 1%, 2%, 5% and 10% trehalose.
  • a blank hydrogel spot which did not contain protein was also included.
  • test solutions were spotted as three pins per sample with two spots per pin onto an amine-coated glass slide.
  • Test protein encapsulated was anti-transferrin, and the system was incubated with Cy3 fluorescent dye-labeled transferrin (Amersham, approximately 0.1 ⁇ g/ml in PBS containing 0.1% Triton X100 (PBST), and 1% bovine serum albumin (BSA)) at 45° with shaking for the indicated times. Following incubation, the slide was washed 2 x 10 minutes in PBST and then imaged using a ScanArray Lite slide scanner. The blank hydrogel spots had no detectable signal, and 0% trehalose had a weak signal. 1% and 2% trehalose were a little more intense, 5% had higher signal yet, and 10% had the strongest signal. These results indicate that the addition of trehalose had a positive effect on the bioactivity of the test antibody in the hydrogel.
  • Panel B Glycerol.
  • Glycerol dissolved as a 20% stock in pH 8.0 sodium borate buffer, was added to the above-mentioned hydrogel formulation containing 3.5% final HYPOL PreMA® G-50, anti-transferrin, bovine IgG, and 5% trehalose, to a final concentration of 0%, 0.5% and 1%, e.g. 0,1.25 ⁇ l and 2.5 ⁇ l of stock glycerol.
  • the Cy3 fluorescent dye-labeled transferrin system was used for assay.
  • mouse IgG was immobilized in 3%, 4% and 5% hydrogel, respectively.
  • BSA was included in as a separate spot as a non-specific binding control.
  • the array was incubated with a solution of rhodamine-labeled rabbit anti-mouse antibody for one hour, then washed.
  • the rabbit anti-mouse antibody bound to mouse IgG antibodies, and the extent of binding was determined by fluorescence at each location using a ScanArray Lite slide scanner. Under identical binding conditions and binding time, the lower percentage hydrogel spots displayed stronger binding signals; this is indicative of a faster diffusion rate of the rhodamine-rabbit-anti-mouse IgG into the hydrogel matrix at these lower percentages.
  • N-hydroxysuccinimidyl active ester (NHS) activated polyethylene glycol (PEG) polymer mPEG-SPA-NHS 5K (Shearwater Corporation) was dissolved in 0.05 M sodium bicarbonate, pH 8.25, buffer to a final PEG concentration of 50 mg/ml.
  • Corning aminosilane slides were used for surface grafting of the polymer.
  • Grace-Biolabs hybridization chambers SA500-3LCLR were used as reaction chambers. To coat the surface, three slides were treated with the PEG solution for 3 hours on a shaker at room temperature (NSH rt), three slides were treated for three hours at 45°C (NSH 45), and an additional slide was treated for 3 hours at 45°C in DI water as a control.
  • single-stranded DNA is first linked to the hydrogel followed by hybridization to create double-stranded binding entities which then are effective to sequester target proteins as schematically shown in FIG. 4A .
  • the resultant double-stranded DNA are then incubated with 1.5 ⁇ g/ml Cy3-labeled bacterial phage lambda repressor ⁇ CI in binding buffer (50 mM Tris.HCl (pH 7.6), 100 mM NaCl, 1 mM CaCl 2 , 0.1 mM EDTA, 0.1 mg/ml BSA, 2.5 ⁇ g/ml poly (dA-dT), 0.05% Tween 20, 1 mM DTT) at room temperature for 2 hours.
  • the Cy3-labeled ⁇ CI is removed at the end of the binding reaction, and the slide is rinsed briefly with binding buffer then deionized H 2 O (dH 2 O) and imaged by a GSI laser scanner.
  • the double-stranded DNA is stained with SYBR Gold (Molecular Probe) according to manufacturer's protocol and visualized by a GSI laser scanner for its total DNA content.
  • double-stranded DNA is pre-hybridized before polymerization and immobilization, which is followed by target protein binding.
  • Double-stranded (ds) DNA biochips can also be made by directly printing 5' amino-modified prehybridized dsDNA. This procedure contrasts with the previous example where a single-stranded DNA was printed, and the cognate oligonucleotide was subsequently hybridized to this printed oligonucleotide to form the binding entity.
  • an estrogen receptor a 53 kD protein, binds as a homodimer to its consensus estrogen response element (ERE).
  • the wild-type ERE sequence differs from the mutant sequence by four nucleotides in a region known to be critical for binding by the receptor.
  • the wild-type sequence is a 32-base oligomer with the sequence 5'-tttacggtagaggtcactgtgacctctacccg-3'.
  • the mutant sequence differs by four oligonucleotides (underlined) and has the sequence 5'-tttacggtagaggtcactgt atgg tctacccg-3'.
  • dsDNA for printing 5 ⁇ l of a 650 ⁇ M stock of each of the amine-linked oligonucleotide of interest and its complementary oligonucleotide are diluted 1:650 (65 ⁇ M final concentration) in 40 ⁇ l DNA hybridization buffer, pH 8 (3xSSC, 5 mM MgCl 2 ) for a final reaction volume of 50 ⁇ l.
  • the reaction product is incubated at 95°C for 10 min and then chilled on ice for 3 min.
  • Ten microliters of this double-stranded DNA is printed within 450 ⁇ m hydrogel spots using a solution consisting of 3.75% polymer, 0.5% glycerol and 50 mM sodium borate buffer, pH 8.0.
  • ER concentration 1.153 ⁇ M was diluted in appropriate binding buffer (10% glycerol, 10 mM HEPES, 30 mM KC1, 0.1 mM EDTA, 0.25 mM DTT, 1 mM Na 2 HPO 4 , pH 7.9) and allowed to bind to the dsDNA for 1 hour at room temperature; a 10-min wash with PBST then followed.
  • the ER-ERE complex was next incubated with a 1:100 dilution of a rabbit anti-ER ⁇ antibody for 1 hour at RT, followed by a 30-min wash with PBST.
  • hydrogel platform can be used as a matrix for anchoring still other binding entities, i.e. antigens.
  • Antibody-antigen interactions are routinely employed in a variety of biological assays, and the ability to anchor either component (antibody or antigen) is a desirable feature in a support.
  • an antigen is anchored within the hydrogel matrix.
  • the protein antigen, human transferrin (0.2 mg/ml), was directly immobilized at different dilutions in 3.3% hydrogel with 5% trehalose, 2 mg/ml BSA onto an amine-coated glass slide. After blocking with 5% non-fat dry milk, the slide was incubated for 1 hour with mouse ascites fluid containing anti-human transferrin at the varying concentrations. After incubation, the slide was washed three times for 10 mins with PBST. The bound, mouse, anti-transferrin antibody was visualized by incubating the slide with Cy3-labeled donkey anti-mouse IgG, followed by laser scanner imaging.
  • antibody-antigen reactions are routinely employed in biological assays.
  • an antibody is anchored within the hydrogel matrix, as opposed to anchoring the antigen in Example 6.
  • Anti-human transferrin, anti-BSA and anti-PSA antibodies (0.4 ⁇ 0.8 mg/ml) were immobilized in 3.3% hydrogel in the presence of 5% trehalose, 2 mg/ml bovine IgG and 0.5% glycerol on amino-silanated glass slides, following the methodology of Example 1.
  • the slides were then incubated at room temperature overnight with Cy3-labeled individual antigens at a concentration of 1 mg/ml in PBST containing 1% BSA. Bound proteins were visualized by laser scanner imaging after an extensive wash with PBST.
  • the presence of labeled target proteins at the sites of the corresponding antibodies on the microarray indicated the retention of functionality of the antibodies in the hydrogel matrix.
  • the ability to support more complex binding interactions may also be a desired feature for the hydrogel matrix.
  • Rat anti-mouse IL-2 monoclonal capture antibody (BD, Pharmingen) was directly immobilized in 3.3% hydrogel with 5% trehalose, 2 mg/ml Bovine IgG on an amino-silanated glass slide, as per the methodology outlined in Example 1.
  • the slide was incubated with diluted culture medium from phytohemaglutinin-stimulated mouse LBRM-33 4A1 cells or unstimulated cells, for one hour with proper mixing at room temperature. After two 15-minute wash PBST washes, the slide was incubated with biotinylated rat monoclonal anti-mouse IL-2 detection antibody (BD, Pharmingen) at room temperature for one hour.
  • Mouse anti-bovine brain calineurin monoclonal antibody (0.4 mg/ml, Sigma), sheep anti-bovine calmodulin antibody (0.2 mg/ml, Chemicon) and control bovine IgG (0.4 mg/ml) were respectively and directly immobilized in 3.3% hydrogel with 5% trehalose and 2 mg/ml bovine IgG onto an amine-coated glass slide, as per the methodology of Example 1.
  • the slide was subsequently incubated with 0.1 mg/ml bovine calcineurin in 20 mM HEPES (pH 7.6), 130 mM KCl, 0.1% Triton X-100,10 ⁇ g/ml polyglutamic acid overnight, after 5% dry milk blocking.
  • Cy3-labeled chicken calmodulin is allowed to bind to the calcineurin-treated slide in the presence of 1 mM CaCl 2 or 5 mM EGTA in PBST, 1% BSA at room temperature for one hour.
  • the bound calmodulin was visualized by laser scanner imaging at the Cy3 excitation and emission wavelengths.
  • a six-fold increase in signal intensity shown at the anticalcineurin antibody location in the presence of calcium as compared to in its absence (i.e. in the presence of EGTA) indicates the ability of the hydrogel matrix to support complex biomolecular interactions involving both proteins and small molecules.
  • the hydrogel matrix is compatible with a wide variety of binding entities and assay formats.
  • binding entities and assay formats In this example, the use of a phosphorylated amino acid within a peptide binding entity is shown.
  • Substrate Amino Acid Sequence 1 insulin receptor fragment NH-ntr-arg-asn-ile-pTyr-gln-thr-asn-tyr-tyr-arg-lys-OH 2 PTP Substrate II NH-asp-ala-asp-glu-PTyr-leu-ile-pro-gln-gln-gly-OH 3 PTP Substrate I Mi-glu-asn-asp-pTyr-leu-ile-asn-ala-ser-leu-OH 4 insulin receptor fragment NH-thr-arg-asn-ile-tyr-gln-thr-asn-tyr-tyr-arg-lys-OH 5 pp60 c-src (521-533) NH-thr-ser-thr-gly-pro-gln-tyr-gln-pro-gly-glu-asn-leu
  • the glass slides were incubated on a rocker in glass slide-staining dishes.
  • the peptide biochip was blocked with 1% BSA in PBS containing 0.1 % Triton X-100 for 60 min at room temperature, followed by overnight incubation at 4°C with biotinylated anti-phosphotyrosine antibody at a 1:2000 dilution in PBST containing 1% BSA. After a 2 times 10 mins wash at room temperature with PBST, the slide was incubated with Cy3-labeled streptavidin at a 1:2000 dilution in PBST containing 1% BSA. Thereafter, the slide was washed 3 times 15 minutes at room temperature in PBST.
  • the slide was air dried and scanned using a GSI Lumonics scanner.
  • the results showed the presence of fluorescent signal at those locations containing phosphotyrosine and not at other locations, including those containing phosphoserine, and indicate that the phosphopeptide, despite isocyanate binding to the hydrogel, retained its appropriate native conformation to allow recognition by the antibody.
  • the previous examples demonstrated the use of the hydrogel matrix to support binding interactions of extended natures (for minutes or hours).
  • the following experiment shows that the matrix also supports transient binding interactions, such as those involving enzymatic activity, as well.
  • a phosphopeptide substrate is anchored within the hydrogel matrix, which then serves as a substrate for an enzyme that removes the phosphate group. Residual phosphates are then detected using the methodology of Example 10.
  • Example 10 Loss of fluorescent signal earlier observed in Example 10 indicated the ability of the phosphorylase enzyme to enter the hydrogel, maintain its biological activity and transiently interact with one or more substrates, i.e. the anchored phosphopeptides.
  • the results show that the LAR-PTPase selectively removes the phosphate group substantially completely from Peptide No. 1 and to a lesser degree on the remaining peptides that contain a pTyr.residue.
  • the YOB-PTPase enzyme substantially completely removes the phosphate group from Peptides Nos. 1, 3, 6, 8 and 13; it removes the phosphate group significantly from Peptides Nos. 2, 7, 10, 14 and 15, i.e. to a greater degree than does the LAR-PTPase for those peptides.
  • the fluorescence results with the various phosphopeptides indicated a preferential specificity on the part of the two phosphorylase enzymes towards certain of the phosphopeptide sequences.
  • Binding entities need not be biological in origin, but a variety of synthetic molecules can be employed as well.
  • a metal chelator is used to anchor a metal ion within the hydrogel matrix where it serves to bind multiple histidine moieties present within a protein molecule.
  • Ni ++ or Cu ++ NTA hydrogel is generated by mixing various amount of nitrilotriacetic acid with HYPOL TM solution and spotted on a glass slide.
  • the polymerized gel spots are washed with 50 mM acetic acid in dH 2 O, charged with 50 mM Cu(NO 3 ) 2 or Ni(NO 3 ) 2 ; they are then washed with 50 mM acetic acid in dH 2 O containing 0.1M KNO 3 (pH4.0) and finally rinsed with dH 2 O 6xHis tagged green fluorescent protein at 10 ⁇ g/ml in PBST containing 1% BSA was added to the slide, which was imaged in PBS by a home-built CCD camera under proper excitation and emission filter, after the removal of free unbound 6xHis-GFP. An increased fluorescent signal is observed that corresponds with increased chelator and indicates that the hydrogel matrix supports the use of small molecules as intermediate binding agents.
  • Alpha-2-macroglobulin is a large plasma protein (mw 800,000) that circulates in the blood specifically to bind to and neutralize proteases, a mechanism which protects the body from excessive protease activity, essentially preventing the body from "digesting" itself.
  • the association between alpha -2-macroglobulin and proteases like trypsin is very strong, and alpha -2-macroglobulin immobilized to agarose beads has been used to affinity-purify trypsin and other proteases.
  • hydrogel microdroplets were spotted onto amine-derivatized glass.
  • the glass slide was first treated with 1% BSA solution in 10 mM sodium phosphate buffer and 150mM NaCl (PBS), pH 7.4, for 2 hours at room temperature to block nonspecific binding sides. Failure to do so would result in some fluorescein-labeled protein binding nonspecifically, thus raising the signal-to-noise ratio.
  • the hydrogel consisted of a prepolymer comprising isocyanate-functional HYPOL TM . Polymerization was initiated with an aqueous solution, and the polymerization kinetics were controlled by pH and temperature.
  • Each hydrogel microdroplet was caused to polymerize at a controlled rate to prevent opacity due to CO 2 gas evolution, forming one cell of the microarray.
  • a first set such of hydrogel cells is loaded with ⁇ -2-macroglobulin using a solution of 50 ⁇ L at a concentration of 5 mg/mL PBS.
  • the high molecular weight of ⁇ -2-macroglobulin limits rapid diffusion into the hydrogel droplet, and the diffusion rate is increased by using a mild electrical current (2.5-5 mV) delivered by a small electrode system.
  • Ferritin is used to provide a negative control protein as it is known not to bind to trypsin. Ferritin is similarly diffused into a second set of hydrogel droplets using the same electrode system and mild electrical current under the same conditions. A third set of droplets is not treated with any protein and serves as an additional negative control. All three sets of droplets are then exposed to FTTC-labeled trypsin for about 15 minutes and washed with 1% BSA-PBS, pH 7.4 for about 5 to 20 minutes. Fluorescence intensities are measured with a CCD camera, and results are shown in Table 2. Table 2. Specific binding of FITC-trypsin to ⁇ -2-macroglobulin Immobilized Protein Flourescence Intensity (au) ⁇ -2-macroglobulin 800 Ferritin 20 No protein 10
  • FTTC-labeled trypsin specifically binds to ⁇ -2-macroglobulin, its natural ligand, within the hydrogel droplets, and that there is little detectable binding activity to either the negative control protein ferritin or to the hydrogel itself.

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Claims (14)

  1. Biopuce d'hydrogel optiquement transparent, comprenant :
    a) un substrat solide ayant une surface ;
    b) une pluralité de gouttelettes d'hydrogel optiquement transparent fixées à la surface du substrat et dans laquelle des protéines différentes sont immobilisées dans des gouttelettes d'hydrogel différentes, lesquelles sont formées à partir d'un polymère à fonctionnalité isocyanate ; et
    c) une protéine immobilisée dans ou sur une telle gouttelette d'hydrogel, protéine qui est efficace pour séquestrer sélectivement une protéine cible ou une autre molécule comparable, et ladite protéine étant choisie dans le groupe consistant en une immunoglobuline, une enzyme, un récepteur, un inhibiteur d'enzyme et un substrat d'enzyme.
  2. Biopuce suivant la revendication 1, dans laquelle l'hydrogel comprend un polymère avec des liaisons uréthanne.
  3. Biopuce suivant la revendication 1 ou 2, dans laquelle l'hydrogel comprend le polyéthylèneglycol, le polypropylèneglycol ou leurs copolymères et dans laquelle la gouttelette d'hydrogel a une épaisseur d'environ 30 µm à environ 100 µm.
  4. Biopuce suivant la revendication 1, dans laquelle ladite protéine est liée par covalence à et dans la gouttelette d'hydrogel par réaction avec des groupes isocyanate, et dans laquelle une proportion égale ou inférieure à 10 % des isocyanates réactifs dans ledit polymère de ladite gouttelette a réagi avec lesdites entités de liaison.
  5. Biopuce suivant la revendication 1, dans laquelle chacune desdites protéines est immobilisée dans l'hydrogel par une interaction avec un agent intermédiaire.
  6. Biopuce suivant la revendication 5, dans laquelle ladite protéine est immobilisée par un premier agent intermédiaire lié à l'hydrogel et un second agent intermédiaire lié audit premier agent intermédiaire.
  7. Biopuce suivant l'une quelconque des revendications 1 à 6, dans laquelle ladite protéine est liée à un chélate métallique qui est immobilisé dans l'hydrogel par un polypeptide contenant de l'histidine à une extrémité de ladite protéine.
  8. Biopuce suivant la revendication 1, dans laquelle ladite protéine est immobilisée physiquement dans ladite gouttelette en résultat de son poids moléculaire.
  9. Biopuce suivant l'une quelconque des revendications 1 à 8, dans laquelle le substrat est optiquement transparent, et dans laquelle le substrat possède des molécules réactives sur sa surface auxquelles l'hydrogel est lié par covalence par certains desdits groupes isocyanates du polymère.
  10. Procédé d'utilisation d'une biopuce pour effectuer une analyse biochimique, procédé qui comprend les étapes consistant à :
    (a) fournir une biopuce d'hydrogel optiquement transparent comprenant un substrat avec une surface à laquelle au moins deux gouttelettes d'hydrogel sont liées, chaque gouttelette ayant une épaisseur d'au moins environ 30 µm et étant constituée de manière prédominante de polyéthylèneglycol, de polypropylèneglycol ou d'un de leurs copolymères, chacune desdites gouttelettes d'hydrogel comprenant une protéine différente immobilisée dans ou sur cette gouttelette, ladite protéine étant choisie dans le groupe consistant en une immunoglobuline, une enzyme, un récepteur, un inhibiteur d'enzyme et un substrat d'enzyme ;
    (b) mettre en contact la biopuce d'hydrogel avec une solution d'analyte, contenant une biomolécule cible dans des conditions de liaison ;
    (c) laver la biopuce d'hydrogel dans des conditions qui éliminent les biomolécules cibles liées non sélectivement et non liées ; et
    (d) détecter la biomolécule cible liée à l'une desdites gouttelettes.
  11. Procédé suivant la revendication 10, dans lequel la liaison de la biomolécule cible entraîne une variation de composition de la protéine, telle qu'un événement de phosphorylation ou un événement de déphosphorylation.
  12. Procédé pour la préparation d'une biopuce d'hydrogel à fonctionnalité isocyanate optiquement transparent comprenant une protéine immobilisée dans ou sur celle-ci, protéine qui est efficace pour séquestrer sélectivement une protéine cible ou biomolécule cible, et ladite protéine étant choisie dans le groupe consistant en une immunoglobuline, une enzyme, un récepteur, un inhibiteur d'enzyme et un substrat d'enzyme, procédé comprenant les étapes consistant à :
    (a) fournir une solution dans un solvant organique d'un prépolymère d'hydrogel à fonctionnalité isocyanate ;
    (b) fournir une solution de ladite protéine ;
    (c) lier par covalence ladite protéine ou un agent de couplage intermédiaire au prépolymère d'hydrogel à fonctionnalité isocyanate par réaction avec une proportion non supérieure à 15 % desdits isocyanates réactifs ;
    (d) déclencher la polymérisation des prépolymères d'hydrogel à fonctionnalité isocyanate dans des conditions qui produiront un hydrogel optiquement transparent ; et
    (e) distribuer le prépolymère d'hydrogel à fonctionnalité isocyanate soumis à la polymérisation sous forme de gouttelettes sur un substrat solide, de telle sorte qu'un polymère d'hydrogel optiquement transparent contenant ladite protéine soit fixé audit substrat.
  13. Procédé suivant la revendication 12, dans lequel une pluralité de gouttelettes d'hydrogel individuelles est dispersée au niveau dudit substrat et est polymérisée pour créer une biopuce comprenant une pluralité de gouttelettes avec des protéines qui sont des agents de capture protéiques, dans lequel des agents de couplage intermédiaires pour ladite protéine sont liés par covalence au prépolymère d'hydrogel à fonctionnalité isocyanate, et dans lequel un groupe desdites gouttelettes est fourni avec une telle protéine et un second groupe de gouttelettes est fourni avec une telle protéine différente en exposant séparément lesdits groupes de gouttelettes à des solutions de protéines différentes pour immobiliser lesdites protéines.
  14. Procédé suivant la revendication 12 ou 13, dans lequel ladite liaison covalente de ladite protéine ou dudit agent de couplage intermédiaire est effectuée simultanément avec la polymérisation en utilisant une solution aqueuse de la protéine ou de l'agent de couplage intermédiaire mélangée à la solution du prépolymère dans un solvant organique.
EP01994529A 2000-10-26 2001-10-26 Biopuces de format tridimensionnel Expired - Lifetime EP1328810B1 (fr)

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ATE421694T1 (de) 2009-02-15
WO2002059372A2 (fr) 2002-08-01
EP1328810A2 (fr) 2003-07-23

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